Program Abstract- Project 2 Currently, there are no licensed human vaccines or antivirals for treating or preventing any encephalitic alphavirus infection. Because epidemics of alphaviruses are sporadic and unpredictable, and endemic disease is common (estimated 10,000 cases annually in the Americas) but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we propose a first of its kind small molecule with prophylactic and therapeutic potential that could be relevant for use both in natural epidemics of V/E/WEEV as well as a deliberate release scenario. In summary, a successful effort will result in a new class of antiviral drugs for treatment of encephalitic alphaviruses; Venezuelan (VEEV), Eastern (EEEV) and Western equine encephalitis viruses (WEEV). Research Project 2 within the U19 Center of Excellence for Encephalitic Alphavirus Therapeutics program will lead, support and manage testing of optimized lead molecules in animal models for safety, toxicity, pharmacokinetics and efficacy. The proposed multidisciplinary efforts are focused on accomplishing studies (Aims 1 and 2) that together with Research Project 1 and 3 will inform selection of the best leads for Technology Readiness Level 5 studies (medicalcountermeasures.gov) mid-way through the program (i.e. Aims 3 and 4). Aim 1 will assess lead optimized molecules for broad spectrum efficacy, PK and dose range finding studies in mouse models and preliminary safety in rat in years 1-3. Aim 2 will assess four lead optimized molecules for therapeutic window, dosing, delay of treatment to define dosing regimen and potential indications for treatment in lethal mouse models of V/E/WEEV in years 1-3. Aim 3 will evaluate lead candidate molecules with favorable criteria in nonGLP and GLP rat and non-human primate (NHP) safety, PK and toxicokinetic studies in collaboration with BASi, a commercial research organization. The pivotal efficacy studies in Aim 4 will evaluate the efficacy of the best lead quinazolinone in two animal species, mouse and cynomolgus monkeys, using dosing regimens defined in the preceding 3-4 years. The Center has two planned meetings with the FDA, one informal meeting mid-way through the program and one formal meeting prior to the efficacy studies in NHP in Aim 4. These studies will contribute to the reports to be readied by our consultants at Leidos.